Method for using naturally occurring gas vesicles as ultrasound contrast agent

ABSTRACT

The present invention generally relates to one or more methods of using naturally occurring vesicles as contrasting agents for ultrasound imaging.

RELATED APPLICATIONS

This application is a continuation-in-part of, and claims priority to,U.S. patent application Ser. No. 10/143,079 that was filed on May 10,2002, which issued as U.S. Pat. No. 7,022,509 on Apr. 4, 2006; which isa divisional of U.S. patent application Ser. No. 09/489,386 that wasfiled on Jan. 21, 2000, which issued as U.S. Pat. No. 6,413,763 on Jul.2, 2002; which is a continuation-in-part of U.S. patent application Ser.No. 08/968,283 that was filed on Nov. 12, 1997, which issued as U.S.Pat. No. 6,036,940 on Mar. 14, 2000; which claims priority to U.S.Provisional Patent Application No. 60/029,432 that was filed on Nov. 12,1996; all of which are incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

This present invention relates generally to microbubbles and, moreparticularly, to naturally occurring gas vesicles. Specifically, thepresent invention relates to methods for using the same as ultrasoundcontrast agents.

Microbubbles, encompassing both natural and synthetic gas-filledmicrocavities, are well known in the art. For example, gas-filledmicrocavities have been employed for enhanced oil recovery, as contrastagents in diagnostic ultrasound, as reagents in in situ bioremediationof contaminated ground water, and as flotation devices for theseparation of minerals.

Microbubbles used heretofore in the art have been synthetic in nature.That is, microbubbles have been produced by methods such as passing airthrough a surfactant solution. One known technique provides a rapid flowof a dilute surfactant solution through a venturi throat through whichgas is emitted to generate the surfactant-stabilized microbubbles.Another example includes employing a triple-barreled jet head thatallows for the simultaneous development of an alginate drop andinjection of an air bubble inside the drop. Depending on the productiontype, these microbubbles have been referred to as microballoons,colloidal gas aphrons, micro gas dispersions, and microfoams.

Although synthetically produced microbubbles are well known in the art,they have several shortcomings. Namely, synthetically producedmicrobubbles lack consistency of size, have poor stability andmechanical strength, and are often biologically incompatible.

Naturally occurring microbubbles, such as gas vesicles, are also known.Many organisms produce and/or employ microbubbles for various biologicalfunctions. Specifically, ecological studies show that manymicroorganisms living in aquatic systems utilize microbubbles asbuoyancy devices. Their importance in providing buoyancy for planktoniccyanobacteria and helping them perform vertical migration in lakes andother aquatic systems has been widely recognized. Additionally, they arepostulated to play a role in light shielding, as well as providing thecell with the ability to alter its configuration to increase cellsurface area as a function of volume.

Among the difficulties in utilizing naturally occurring microbubbles forcommercial purposes is the fact that it is difficult to harvest them.For example, in typical industrial microbial fermentations, cells arecollected by either filtration or centrifugation. Filtration involveslarge pressure gradients or mechanical forces that tend to collapse thegas vesicles, and centrifugation is inefficient because thevesicle-bearing cells often have densities very close to that of water.Further, where centrifugal force is strong enough to achieve efficientcell collection, such forces often destroy the gas vesicles. It is alsodifficult to sterilize them so that they can be kept stable againstmicrobial and enzymatic attack.

Biological systems often produce gaseous compounds as by-products oftheir metabolism. When these compounds accumulate, they can inhibit thegrowth, product synthesis and even survival of the biological system. Acommon example of such a gaseous compound is carbon dioxide. If it isnot removed effectively, carbon dioxide accumulation can negativelyaffect plant cell cultures, insect cell cultures, animal cell cultures,and microbial fermentations. Furthermore, the low shear requirements ofmany types of cell culture lead to poor gas-liquid interfacial transferand, consequently, potential accumulation of inhibitory or toxic gaseousmetabolic by-products.

Thus, there is a need in the art to overcome the shortcomings ofsynthetically produced microbubbles. Further, there is a need in the artto overcome the difficulties in harvesting naturally occurringmicrobubbles and utilizing such microbubbles for commercial purposes inlieu of synthetic microbubbles. Still further, there is a need in theart to overcome the difficulties associated with the removal of gaseousmetabolic byproducts from biological systems.

SUMMARY OF THE INVENTION

This present invention relates generally to microbubbles and, moreparticularly, to naturally occurring gas vesicles. Specifically, thepresent invention relates to methods for using the same as ultrasoundcontrast agents.

The present invention also relates to process of using semi-syntheticgas vesicles as a contrasting agent, comprising: injectingsemi-synthetic gas vesicles in the vicinity of a biological target,wherein the vesicles associate with the target and are operable toreflect ultrasonic acoustic waves; subjecting the target to ultrasonicacoustic waves; collecting a portion of the reflected ultrasonic waves;converting the ultrasonic waves to an electrical signal; transmittingthe electrical signal to a computer, wherein the computer is programmedto convert the transmitted electrical signal into an image of thetarget; and displaying the image.

The present invention also relates to an ultrasonic contrast agent,comprising: Gas vesicles, wherein the vesicles are crosslinked.Furthermore, the present invention relates to a process of making anultrasonic contrasting agent, comprising: providing gas vesicles;contacting the gas vesicles with a crosslinking agent; crosslinking thegas vesicles with the crosslinking agent; and recovering the crosslinkedgas vesicles. Finally, the present invention relates to an ultrasoniccontrast agent made by the foregoing process.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are a schematic side view and end view, respectively, ofa cyanobacterium gas vesicle;

FIGS. 2A and 2B are the effect of nitrate (NO₃ ⁻) and light intensity,respectively, on growth of cyanobacteria;

FIG. 3 is profiles of cell concentration, cell density, specific gasvesicle content and percent buoyant filaments from a batch culture ofAnabaena flos aquae;

FIG. 4 is a 20-hour batch flotation study for evaluating the effect ofculture growth phase on buoyancy;

FIG. 5 is pressure collapse curves for gas vesicles of Anabaena flosaquae;

FIG. 6 is the effect of glutaraldehyde treatment of gas vesicles onexposure to 2.5% (w/v) SDS;

FIG. 7 is an apparatus for evaluation of oxygen supply and carbondioxide removal capacity of gas vesicles in animal cell culture; and

FIG. 8 is profiles of glucose concentration and glucose utilizationrates in a perfusion run with DMEM containing 1% serum and latersupplemented with 1.8% gas vesicles.

DETAILED DESCRIPTION

The present invention involves the production, modification andapplication of naturally occurring gas-filled microcavities. Forpurposes of this disclosure, these gas-filled microcavities will bereferred to as gas vesicles, or in the case where they have beenmodified, semi-synthetic gas vesicles. Naturally occurring gas vesiclesare those preferably found within living organisms. Most preferably,these gas vesicles are located within the cell or cells of livingorganisms and are often found as a group of vesicles or a vacuole. Gasvesicles can occupy up to about 10% of the cell volume. The best knownexamples of gas-vesicle-containing cells are those of cyanobacteria,halobacteria, and phototrophic bacteria.

As depicted in FIGS. 1A and 1B, gas vesicles 2 are proteinaceousstructures that are usually shaped as hollow cylindrical tubes 4 closedat each end by a hollow conical cap 6. These structures are typicallyhydrophobic, thereby preventing the penetration of water and other polarliquids by surface tension, but are permeable to gas molecules. Due totheir permeability, the interior of the vesicles become filled with thegas or gases contained in the surrounding environment; typically, thegases are dissolved in a surrounding aqueous environment. Further, it isbelieved that these structures gain their dispersability in aqueousmedia from hydrophilic proteins attached to the outer surfaces of thegas vesicles.

Typically, the walls of naturally occurring gas vesicle structures arequite rigid and withstand considerable hydrostatic pressure with littlechange in overall volume. The various sizes of the reported gas vesiclesrange from about 340 to about 750 nm in length and from about 60 toabout 110 nm in width, depending on their biological sources. The wallthickness of such gas vesicles is typically about 2 nm. The submicronsize of gas vesicles and their inherent strength and stability give thema substantial advantage over synthetically produced microbubbles.

It should be understood that a variety of naturally occurring gasvesicles can be employed in the present invention. For example, they canbe obtained from various procaryotes, including cyanobacteria such asMicrocystis aeruginosa, Aphanizomenon flos aquae and Oscillatoriaagardhii; phototropic bacteria such as Amoebobacter, Thiodictyon,Pelodictyon, and Ancalochloris; nonphototropic bacteria, such asMicrocyclus aquaticus; and archaea, such as Haloferax mediterranei,Methanosarcina barkeri, Halobacteria salinarium. Preferred procaryotesare filamentous, for ease of separation, and have high vesicle content,by volume, for greater gas delivery or removal capability. Therefore,Anabaena flos aquae, where the vesicles comprise about 10% of the volumeof the cell, is a preferred source of naturally occurring gas vesicles.

To utilize naturally occurring gas vesicles in a gas delivery or removalsystem, one embodiment of the present invention entails harvesting andcollecting cells that have naturally occurring gas vesicles therein. Aspreviously mentioned, known methods for collecting cells containingnaturally occurring gas vesicles employ methods that place too muchstress on the gas vesicle. These considerations, however, have not beenproblematic inasmuch as methods or techniques of collecting naturallyoccurring gas vesicles have been employed simply for small-scale,laboratory studies, not for future uses as contemplated by the presentinvention.

The organisms from which the gas vesicles of the present invention areobtained can be cultivated either in open waters, such as lakes andponds, or in sterile bioreactors having well controlled processconditions. Ultimately, the intended end usage of the gas vesicles playsa significant role in the initial environment that the organism shouldbe cultivated in. For example, uncertain contamination that can beexpected in open water cultivation often prohibits the use of gasvesicles obtained therefrom for biological and medical applications. Itis therefore preferred to obtain naturally occurring gas vesicles fromcells cultured in bioreactors. Furthermore, within bioreactors, theprocess conditions can be controlled and optimized to achieve desirablecell and gas vesicle productivity. This is especially true where certaincharacteristics of the cell culture are desirable for cell collection.

The preferred method of harvesting cells containing gas vesiclesincludes manipulating environmental and cellular conditions to maximizeflotation. With reference to FIG. 2A, it is preferred to culture thepreferred cells employed in the present invention in an environmentcontaining nitrate. As can be seen in the data plot of FIG. 2A, thegrowth of cyanobacteria with nitrate has a higher exponential growthrate and a reduced lag phase. Significantly, it has been found that thecultures are most buoyant and float most rapidly during theirexponential growth stage.

It has been found that for the preferred species of bacteria, which arenitrogen-fixing bacteria, a usable nitrogen source in the medium aids incell growth, thereby enhancing the yield of gas vesicles. If the onlynitrogen source in the medium is nitrogen gas (N₂), it is believed thatthe cells must consume large quantities of energy to fix nitrogen,thereby interfering with cell growth. Therefore, a preferred nitrogensource is one that can provide nutrition to the cells while requiringless energy to metabolize than does nitrogen gas (N₂).

Although one skilled in the art will realize that many sources ofnitrogen are feasible, the preferred sources of nitrogen are ammoniumand nitrate ions, preferably added to the medium as a salt. These ionsare most preferred because of their low toxicity and low expense. It hasbeen found that bacteria cells grow faster in the presence of ammonium,provided the concentration is kept low. Above about 10 mg NH₄ ^(+—)N/L,the ammonium ions become toxic to the cells. Therefore, it is mostpreferred that from about 3 to about 10 mg NH₄ ⁺—N/L is provided to thecells by frequently adding small quantities of ammonium salt. Althoughthe cells do not grow as fast with nitrate as with ammonium, higherconcentrations of nitrate can be added to the cells without being toxic.Therefore, the addition of one batch of from about 10 to about 100 mgNO₃ ⁻—N/L can be used.

It has also been found that cell cultures are more likely to becomebuoyant and float under dark conditions wherein the cells consumeintracellular carbohydrates and become less dense. Therefore, it ispreferred to collect cells from the surface of a medium in the darkstage once the cells have had sufficient time to float to the surface.Although the minimum time one must wait depends on the distance thecells must travel to reach the surface, generally about 5 to about 15hours is ample. Keeping the cells in darkness for more than about 24hours should be avoided, however, because the cells will begin to dieand sink at about this time.

Generally, cell culture growth undergoes an exponential stage, followedby a linear stage and then a stationary or plateau stage. The maximumconcentration achieved depends on the growing conditions. One importantgrowing condition is the amount of light the cells are exposed to duringgrowing. It has been found that the best growth occurs when cells areexposed to at least about two watts per square meter; however, at anintensity greater than about 2 klx, the vesicles start to collapse,presumably due to an increase in intracellular, or internal, osmoticpressure. Since the light is most intense at the directly illuminatedsurface of the medium, the ideal surface light intensity for culturesthat are mixed is greater than for cultures that are not mixed.Therefore, an example of one preferred bioreactor is one containingabout 5 liters of culture medium stirred at a rate of about 200 rpmunder a surface light intensity of from about 1.1 klx to about 2.1 klx.

Accordingly, the cells are preferably cultured in continuous light andplaced in the dark when flotation is desired. The light is preferablyfluorescent due to its inexpense and availability, but it could also befrom other sources of white light or colored light. If colored light isused, it is preferable to use light at about 430 nm and/or about 680 nmbecause it is believed that these are the peak absorption wavelengths atwhich light is harvested by the chlorophyll of the cells. Preferably,the light exposure is continuous because it is believed that the cellsconsume food reserves in the dark and cell growth is slower.Accordingly, a method of collecting cells according to one embodiment ofthe present invention entails placing the cells, preferably during theirexponential growth stage and preferably in a dark environment, so as tocause the cells to float to the surface of the aqueous medium in whichthey are contained. These cells can then be physically removed from theaqueous medium's surface by suction. One skilled in the art willappreciate, however, that there are other means for physically removingor collecting the cells.

The collection methods described hereinabove can be further improved byinducing flocculation. Flocculation, or aggregation, of the culture togenerate large flocs of cells can amplify the effect of densitydifferences and shorten the process time significantly. While thedensity difference is the driving force for particle flotation orsettlement, the speed of the particle movement in the process isproportional to the square of the particle size, deviating significantlyonly at high cell concentration. Flocculating agents, such aspolyethylenimines, polyacrylamides, polyamine derivatives, ferricchloride, and alum, can be added to induce the flocculation process. Theskilled artisan will realize, however, that additional flocculatingagents, or combinations thereof, could also be used.

According to another embodiment of the present invention, cells can becollected in a non-exponential stage. Indeed, it is desirable to harvestat a later stage for higher cell and, thus, higher gas vesicleconcentrations. Since natural buoyancy is diminished in thepost-exponential stages of growth, flotation can be achieved bydissolved gas flotation. A liquid, preferably aqueous, is firstgas-pressurized to a high pressure to dissolve large amounts of gas init, preferably about 50 to about 80 atm. One skilled in the art,however, will realize that higher or somewhat lower pressures could alsobe used. The liquid is then injected into a medium containing theculture under lower pressure, preferably ambient pressure. The suddenpressure drop causes many very fine bubbles to form because the mediumbecomes over-saturated with gas. The tiny bubbles float to the medium'ssurface, carrying with them the filaments that they encounter in theirpath. With this technique the flotation of the cells is achievedprimarily due to the buoyancy of the gas bubbles. It is thereforesubstantially independent of the cell buoyancy and can be applied to theculture harvested at any stage. It should also be noted that the type ofgas used is not critical because the gas is merely a carrier. Readilyavailable gases such as air, oxygen, nitrogen, or carbon dioxide, forexample, could be employed.

Following collection of the cells, the gas vesicles must be removed fromthe cells. Removal generally requires lysis of the cell wall, which isachieved by placing the cells in a hypertonic sucrose solution, such asa 0.7 M sucrose solution. Within such an environment, the cells shrinkunder osmotic pressure and eventually rupture. After the cells have beenruptured, the extracellular gas vesicles can be separated from the wastecell material by filtration or centrifugation methods. It should beappreciated that because there is a large density difference between thegas vesicles and waste cell material and because of the release ofintracellular osmotic pressure, techniques such as centrifugation arenow useful.

Once extracted and separated from the cell material, the gas vesiclesare preferably modified to improve their stability and mechanicalstrength. Specifically, the present invention provides for crosslinkingof the protein surface of the naturally occurring gas vesicles. As aresult, the gas vesicles have greater tolerance for fluctuations intemperature or pressure and greater resistance to chemical, microbial orenzymatic agents. It has also been found that crosslinking of theprotein surface also serves to sterilize the gas vesicle suspension forlong term storage. Most, if not all, crosslinking agents contemplated bythe present invention crosslink all proteins found in the medium.Therefore, the enzymes of microorganisms present in the medium alsobecome crosslinked, thereby killing the microorganisms. Thiscrosslinking of the protein surface further serves to render the gasvesicles more suitable for biological and medical applications.

Crosslinking agents that can be employed include any bifunctionalcrosslinking agent. Examples of such crosslinking agents includebisdiazobenzidine, N,N′-ethylene bismaleimide, hexamethylenediisocyanate, toluene diisocyanate, hexamethylene diisothiocyanate,N,N′-polymethylene bisiodoacetamide, and preferably glutaraldehyde.

Although crosslinking is preferred, gas vesicles can be strengthened orstabilized by other methods, such as interfacial polymerization orencapsulation. Interfacial polymerization is based on diffusionretardation of the polycondensation reaction of two highly reactivedifunctional monomers. Two monomers, one inside the gas vesicle and oneoutside, meet at the interface, the gas vesicle coat, and the fastpolymerization reaction forms a thin polymer film at the interface. Acrosslinking agent can be used to further increase the strength of thepolymer film.

Encapsulation can be achieved by phase separation (coacervation) or byliquid drying methods. Briefly, for coacervation, a polymer is dissolvedin an organic solvent and precipitated onto the vesicles by addinganother organic solvent that is miscible with the first but does notdissolve the polymer. If the temperature and the amount of organicsolvent added are varied, the polymer solution can be separated into twophases containing low and high concentrations of polymer.

For liquid drying, gas vesicles can be suspended in a polymer solutionwhose solvent is water-immiscible. The suspension is then dispersed inan aqueous solution and dried by warming under a vacuum, for example.The removal of organic solvent leaves a thin coating of thewater-insoluble polymer on the surface of the gas vesicles.

Interfacial polymerization and coacervation both add strength to thevesicles. These methods are generally not preferred when gas delivery orremoval is the intended application, however, because the surfacecoatings slow down the mass transfer of gas across the vesicle wall.

Furthermore, it should be appreciated that the crosslinking of theproteinaceous surface improves the stability of the gas vesicle evenunder the stripping action of SDS. This increased stability issignificant because surface-active substances are typically encounteredin most pharmaceutical and biological manufacturing processes. It alsoshould be appreciated, however, that this further step of crosslinking,although preferred, is optional.

The amount of crosslinking agent employed is a function of the vesicleconcentration in the solution and the type of crosslinking agent. Forexample, it has been found useful to employ from about 0.001 to about 5%(w/v), preferably from about 0.05 to about 0.15%, and more preferablyfrom about 0.08 to about 0.12% glutaraldehyde in a lysate or othersuitable solution. It is preferable to crosslink the gas vesicles beforeharvesting—while in the lysate—in order to maximize the yield of intactgas vesicles.

It is also preferable to remove excess crosslinking agent from the gasvesicles. In a preferred embodiment of the present invention, whereglutaraldehyde is employed, removing excess glutaraldehyde is especiallyimportant because it is a toxic substance that would preclude employingthe gas vesicles in biological or medical purposes if such residualcrosslinking agent were not removed. One method for removing excesscrosslinking agent is by successive dialyses of the treated gas vesiclesuspension.

Once the naturally occurring gas vesicles of the present invention havebeen harvested and, optionally, modified, the gas vesicles may then befilled or loaded with a gas to be carried. The gas is loaded by allowingthe gas vesicles to equilibrate in the presence of the gas. It should benoted that in a preferred embodiment, gas transfer across the very thinproteinaceous coat of the gas vesicles occurs quickly relative to thediffusion of the gas in the thicker surrounding aqueous or liquidmedium. Therefore, the vesicle coat is typically not the rate-limitingstep of gas transfer for either the loading or releasing stage.

One preferred use of the gas vesicles of the present invention includesusing the gas vesicles as oxygen carriers in biological applications.For example, the gas vesicles, having oxygen loaded therein, can be usedto deliver oxygen to cells, whether the cells are isolated or in theform of tissues or organs. This is significant inasmuch as oxygen cannotbe directly delivered to animal cell cultures at high rates usingtraditional methods, such as mechanical mixing, because animal cells arevery sensitive to shear forces.

A similar preferred use of the gas vesicles of the present inventionincludes using the gas vesicles as carriers for the removal ofinhibitory or toxic metabolic gases, such as carbon dioxide, nitricoxide, ammonia, volatile alcohols such as methanol and ethanol, andvolatile organic acids such as formic acid and acetic acid, inbiological applications. For example, the gas vesicles, having a gaswith a low partial pressure of carbon dioxide loaded therein, can beused to remove carbon dioxide from cells, whether the cells are isolatedor in the form of tissues or organs. As mentioned above, this issignificant inasmuch as carbon dioxide cannot be directly removed fromcertain cell cultures such as animal and insect cells and tissues athigh rates using traditional methods, such as mechanical mixing. Evenmild shear such as that associated with bubbling, may cause cell deathor alteration of the normal activity of the cells. Such a low shearenvironment, however, leads to poor gas-liquid interfacial transfer and,consequently, potential accumulation of the inhibitory gaseousmetabolites.

Furthermore, delivery of one gas and removal of another may naturallyoccur simultaneously. For example, if gas vesicles are loaded withoxygen, the vesicles can deliver oxygen and remove carbon dioxide at thesame time. It is possible, however, for one gas to be delivered withoutremoving another and vice versa. For example, gas vesicles can be loadedwith gas that has a partial pressure for oxygen that is equal to that ofthe medium, but has a lower partial pressure for carbon dioxide than themedium. In such a case, carbon dioxide will partition into the vesiclesbut oxygen will not diffuse out of the vesicles because of the relativepartial pressures of each gas inside and outside the vesicles. A gaswill diffuse into or out of the vesicles only to the extent necessary toequalize the partial pressure of that gas inside and outside thevesicles.

Use of gas vesicles may be especially advantageous in biological systemsand processes employing high cell densities. High cell densityintensifies the generation rates of undesirable metabolic compounds andis often associated with high viscosity of the culture broth because ofthe large amounts of cells in the suspensions. Membrane bioreactors suchas hollow fiber reactors and other cell immobilization techniques aretypically used to retain cells and achieve high cell densities. Thesesystems do not allow direct contact between culture broth and the freegas phase to remove metabolic gases effectively.

Furthermore, because oxygen has low solubility in aqueous media, lowoxygen and high carbon dioxide concentrations are often encountered inanimal cell cultures. These concentrations are sometimes at levels whichdo not allow complete aerobic functioning of the cells. This directlyinfluences the maximum cell concentration achievable and thereby resultsin a low product titer and a low process productivity. To overcome theseproblems, the present invention provides a method whereby oxygen-filledgas vesicles are introduced into the cell or tissue environment toincrease the oxygen content and/or to decrease the carbon dioxidecontent thereof. It is envisioned that for most oxygen-delivery ormetabolite-removal uses, the highest achievable concentration of gasvesicles is desirable for maximum oxygen delivery. Although expensiveand time-consuming, concentrations up to about 30% (v/v) are possiblebefore being limited by high viscosity. It is envisioned that for mostoxygen-delivery or metabolite-removal applications, a gas vesicleconcentration between about 0.01 and about 10% (v/v) is effectivewithout undue expense. The cost, however, is also dependent on whetherit is feasible or desirable to recover or reuse the gas vesicles at ahigh concentration. Furthermore, for other uses, such as imaging, lowerconcentrations may suffice.

To demonstrate the practice of the present invention, the following wasperformed. It will be appreciated that the present invention, whileencompassing all of the following example(s), is not necessarily limitedthereto, and that other embodiments and characterizations may fallwithin the scope of the invention.

Example

The filamentous gas vacuolate cyanobacterium strain Anabaena flos-aquaeCCAP 1403/13f was obtained from the Institute of Freshwater Ecology,Windermere, UK. The medium used was as follows: 39 mg/L K₂HPO₄, 5 mg/LNa₂CO₃, 37 mg/L MgSO₄.7H₂O, 9 mg/L CaCl₂.2H₂O, 12 mg/L Na₂EDTA, 1 mg/LFeSO₄, 0.51 mg/L MnSO₄.4H₂O, 0.046 mg/L (NH₄)₆Mo₇O₂₄.4H₂O, 0.71 mg/LH₃BO₃, 0.55 mg/L ZnSO₄. 7H₂O, 0.020 mg/L CuSO₄.5H₂O, 0.012 mg/LCo(NO₃)₂.6H₂O, and 600 mg/L NaNO₃. The original strain was maintained byregular subculturing in 40 mL glass vials and using the original medium.The original medium did not contain any NaNO₃.

Cultures were grown in glass columns bubbled with air from aquariumpumps and passed through humidifiers before being dispersed bydispersion stones placed at the bottom of the vessel. Columns of threedifferent sizes with diameters of 5, 6.5 and 14 cm, heights of 27, 35and 42 cm and with operating culture volumes of 0.5. 1.0 and 5.0 liters,respectively, were used. The temperature of the culture was maintainedat 19° C. by placing the 0.5 and 1.0 L vessels in a refrigerated waterbath set at that temperature and by passing water from the bath throughcooling coils provided in the 5.0 L column. Sylvania 20 W cool whitefluorescent lamps were placed around the photobioreactors to provide forillumination. The average surface intensity of the light, measured usinga Lutron LX-101 lux meter, was between about 0.2 to 2.1 klx.

FIG. 2B shows that increasing the light intensity from 1.1 klx to 2.1klx after 20 days, as indicated by the arrow, caused additional cellgrowth before plateauing at a higher cellular concentration. As anaside, it should be noted that the cell culture shown in FIG. 2B,although containing nitrate as in FIG. 2A, was grown in a much largerbioreactor, had a higher initial inoculant, and had a higher lightintensity (growth in FIG. 2A was under a light intensity of 0.3 klx).This accounts for the higher cell concentration shown in FIG. 2Brelative to that shown in FIG. 2A.

The starting pH of the media was maintained at 7.5 and was followedduring the fermentation using an Orion pH meter (model 420A). The pHelectrode was calibrated in the range of 7 to 10, with appropriatebuffer solutions, prior to the measurement of pH. The measurements weremade within 10 minutes of taking the samples in order to minimize theeffect due to any physiological changes that the cells may undergo.

A calibration curve between the optical density and the cell dry weightwas generated. Optical density measurements on culture samples were madeusing a Perkin-Elmer UV/VIS Spectrophotometer (model Lambda 3B) at awavelength of 685 nm. Appropriate dilutions were made if the opticaldensities were beyond the Beers law range. Dry cell weight was measuredby first collapsing the gas vesicles in a 50 mL sample of known opticaldensity, centrifuging the cells, withdrawing the clear supernatant,washing the cells by resuspending and recentrifuging in distilled water,resuspending again in distilled water and drying the cells at 90° C. for24 hours.

Exponentially growing cells with an optical density of about 1.0 to 1.5had ideal flotation characteristics and floated rapidly (within 15hours) to the surface, when the culture was left undisturbed in thedark. Flotation was achieved by placing the culture in graduatedcylinders in the dark. The cylinders varied in size from 25 mL to 5.0 L.Flotation was quantified in terms of the rate of rise of filaments tothe surface of the undisturbed culture and by the clarity of thesubnatant. This phenomenon can be explained by looking at the profilesfor cell density, gas vesicle content and the % buoyant filaments.

With reference to FIG. 3, the increased buoyancy and decreased filamentdensity of cells during their exponential growth stage is represented.Also represented is the specific gas vesicle content, i.e., the gasvesicle concentration per unit cell, which remains relatively constantat various growth stages. The initial fluctuation shown in the graph isdue to the experimental uncertainties associated with the measurement ofvery low cell concentrations.

FIG. 4 demonstrates the advantage of collecting the cultured cellsduring their exponential growth stage by the flotation method. Atvarious stages of cell growth, flotation was achieved by placing thecells in darkness for about 20 hours. For the cells at the exponentialgrowth stage, it is clear that as the carbohydrate concentration withinthe cell drops, so does the cellular density.

Cell lysis was done by osmotic shrinkage of the cells when placed instrongly hypertonic sucrose solutions. The cell wall is placed undertension and tends to rupture. Osmotic shrinkage has been tried beforebut our method was modified to improve the recovery of gas vesicles.Instead of adding an equal volume of 1.4M sucrose solution to theconcentrated cells and reducing the final sucrose concentration to 0.7M,which was done previously by others, we modified the method in order toreduce the volume for the centrifugation step. Theharvested/concentrated cell suspension was divided into two equalvolumes. To one half, crystalline sucrose was added to a concentrationof 1.4M. It was dissolved by rapid mixing (gentle stirring) with handafter which the other half was added to give a final sucroseconcentration of 0.7M. This way we reduced the operating volume by half,without losing any gas vesicles, and was found to be very beneficial.Secondly we eliminated the use of magnetic stirring as recommended byothers as we found that it led to gas vesicle loss due to grindingaction. Lysis was complete after the suspension was left at roomtemperature for 2 hours, during which time the flask containing thelysate was gently swirled every half hour for 1 minute. On completion oflysis (after 2 hours) the lysate was placed in the refrigerator and waskept there at all times prior to gas vesicle isolation.

FIG. 5 shows that the stability of the gas vesicles, when isolated fromthe cell, is improved. This is presumed to be due to the fact thatintracellular turgor pressure is absent after lysis. Intact gas vesicleshave a very low density (ca. 100 kg m⁻³) and despite their small sizefloat up more rapidly than any other cell components. The process can beaccelerated by centrifugation, though this may generate sufficientpressure to cause their collapse. Since maximum pressure generated is atthe base of the centrifuge cup, the highest possible acceleration isgiven by a=p/hp, where p is the minimum critical collapse pressure ofthe gas vesicles (i.e., the lowest pressure at which there is any gasvesicle collapse), h is the height of lysate in the cup, and p is thedensity of the lysate.

The lysate was centrifuged at 1500 rpm in test tubes filled to a heightof 5 cm (ca. 8 mL/tube) for 8 hours. This was based on a minimumcritical collapse pressure of 2 atmospheres. The lysate was layered ontop with a 5 mm layer of phosphate buffer which helped rinse thevesicles. The gas vesicles were collected from the top using a pasteurpipet with the tip of the pipet held just in contact with the meniscusat the wall of the tube. The gas vesicles were next passed through a 1.4micron filter to remove any contaminating cells that might have floatedup with the gas vesicles and then resuspended in distilled water andrecentrifuged.

Suspensions of both isolated gas vesicles and gas vacuolate cellsscatter light strongly. Most of the light scattering is due to the gasspace rather than the enclosing gas vesicle wall; the amount of lightscattered decreases by 98% or more when gas vesicles are collapsed bypressure. The scattered light can be measured directly in a pressurenephelometer to give a relative measure of gas vesicle content.

A Hach Model 2100 A Turbidimeter was used for making pressurenephelometric measurements. The nephelometer was first calibrated usinggiven standards. A thick-walled glass tube filled with 7 mL of eitherthe gas vesicle or the gas vacuolate cell suspension was next insertedinto the nephelometer with the pressure-tight coupling attached. A lidwas then used to completely cover the tube so that there was nointerference due to external light falling on the sample. With theoutlet valve closed, the inlet valve was slowly opened and the pressureraised in steps of 10 psi (about 0.7 atm). The light-scattering reading(Nephelometric Turbidity Unit, NTU) falls as gas vesicles are collapsedunder pressure. The reading was left to stabilize at each step for a fewseconds before proceeding to the next. No further decrease in NTU is anindication that all gas vesicles have collapsed. On releasing thepressure there is no increase in the turbidity, implying theirreversible collapse of gas vesicles.

The volume of gas vesicle gas space in a sample was determined from thecontraction caused by application of pressure in a capillary compressiontube. The precision bore capillary of 200 micron diameter (31.4 nL/mm)and the attached reservoir (ca. 2.5 mL) are filled with either the gasvesicle suspension or the gas vacuolate cell suspension depending on theexperiment. The reservoir end is then closed with a wetted nylon stopperwhich makes the socket air tight. The tube is then carefully checked forany air bubbles that might be trapped inside, in which case theprocedure is repeated. The capillary tube is then placed inside thethick-walled glass pressure tube which is maintained at 18° C. bypassing cooling water through the outside water jacket. The reservoirend of the assembly is then sealed by using a threaded plug. Air issucked into the top centimeter or two of the capillary as the samplecools down. The assembly is next tilted slightly and the pressure tubecompletely filled with water. At this point water gets pulled into thecapillary resulting in a trapped bubble. The other end of the pressuretube is connected to a compressed air cylinder with a pressure gauge.The assembly is then mounted on a movable steel base which is connectedto the free end of the sliding arm of a vernier calliper. A microscopefixed on top of the assembly focuses on the meniscus of the air bubblecloser to the reservoir end.

The meniscus is allowed to stabilize and the reading on the verniernoted (a). The chamber is next pressurized to 150 psi (ca. 10 atm) uponwhich the bubble contracts. The meniscus reading (b) is again notedafter it stabilizes. Two more readings are taken on the sample: oneafter releasing the pressure (c) and the other upon repressurizing to150 psi (d). Gas vesicle volume can be calculated from the value[(a−b)−(c−d)]. All measurements were made in inches from the vernierscale.

The density of cyanobacteria was measured by isopycnic banding oncontinuous gradients of the silica sol, Percoll, which is a registeredtrademark of Pharmacia Fine Chemicals. Gradients were formed bycentrifuging 10-mL samples, consisting of 2× concentrated cell-freeculture medium (5 mL) and Percoll (5 mL), at 32000 g for 1 hour at 5° C.in a fixed angle rotor. This gave a smooth gradient ranging in densityfrom 1000 to 1200 kg m⁻³. The gradient was overlaid with 1 mL of theculture and recentrifuged at 100 g for 20 minutes in a swing-outcentrifuge. The filaments formed a visible isopycnic band. The densityof this band was determined by using density marker beads which wereused to calibrate the gradients. They were used as an external standard,i.e., in a centrifuge tube run simultaneously with the one containingthe experimental sample.

Crosslinking of gas vesicles was attempted by treating gas vesiclesuspensions with glutaraldehyde. Different concentrations ofglutaraldehyde were tested, ranging from 0.001% (w/v) to 5% (w/v). 0.200mL of a concentrated gas vesicle suspension were added to 7 mL of 0.1 Mphosphate buffer. These values were used in order to obtain an initialnephelometric turbidity reading of about 35 units, which was found to beideal in terms of the range of turbidity available to generate pressurecollapse curves. Glutaraldehyde was next added from a 25% (w/v) solutionto different vials containing the gas vesicle solution to obtainconcentrations ranging from 0.001 to 5% (w/v). One vial was kept as acontrol with no glutaraldehyde. The reaction was allowed to run for 3hours. After this treatment, pressure collapse curves were generated forall the samples using the pressure nephelometer.

With reference to FIG. 6, the improved stability provided bycrosslinking of the proteinaceous surface of the gas vesicle isdepicted. As can be seen, at about 4 atm of applied pressure, only about20 to about 30% of the gas vesicles collapsed after being crosslinked byglutaraldehyde in the absence of the surfactant sodium dodecylsulfate(SDS). Detergents like urea (6M) and SDS (2.5% w/v) have been found tosignificantly weaken gas vesicles due to the stripping of GvpC.Experiments were done to see if glutaraldehyde treatment to crosslinkthe gas vesicles reduced protein stripping by these detergents. Thiswill provide a measure of the extent of crosslinking achieved.

0.4 mL of concentrated gas vesicle suspension were mixed with 3.5 mL of0.1 M phosphate buffer at pH 8.0. Glutaraldehyde was added to make aconcentration of 0.1% (w/v) and the treatment was done for 3 hours. 3.5mL of 12 M urea was then added to the treated samples. Since SDS wasfound to precipitate in 0.1 M phosphate buffer, nephelometric turbiditymeasurements could not be made on samples containing SDS. As a result,0.4 mL of concentrated gas vesicle suspension were mixed with 3.5 mL ofdistilled water and the pH adjusted to 8.0. 3.5 mL of 5% (w/v) SDS weremixed with an equal volume of the gas vesicle containing sample. Gasvesicle collapse curves were generated by pressure nephelometry on thesamples rinsed with SDS.

The ability to deliver oxygen and to remove carbon dioxide wasdemonstrated using a perfusion culture of Vero cells according to theexperimental set up represented generally by the numeral 10 in FIG. 7.Vero cells are kidney cells of the African green monkey having anaverage cell size of about 25 μm. Vero cells are an anchorage-dependentcell line and need a substratum upon which to proliferate. They weretherefore grown on glass microcarrier beads 12 with an average diameterof about 180 μm. Beads 12, having attached Vero cells, were packed intoa thin glass column 14 having a diameter of about 1 cm and a volume ofabout 5 mL. Using a set of pumps 16 a and 16 b, a culture medium 18 wasperfused back and forth through tubing 20 a and 20 b and column 14 at arate of 0.56 mL per minute from reservoirs 22 a and 22 b, sealed bystoppers 23 a and 23 b, respectively. An on/off controller 24, havingtwo independently programmable timers, controls the perfusion. Pump 16 acontrols the clockwise flow of medium 18 by pumping air clockwisethrough conduits 26 a and 26 b. Likewise, pump 16 b controls thecounterclockwise flow of medium 18 by pumping air counterclockwisethrough conduits 28 a and 28 b.

The cell growth profile was followed with time, adding glucoseperiodically to maintain the concentration of glucose between about 1000and about 5500 mg/L. The glucose utilization rate, which is proportionalto cell concentration, was used as a proxy for cell concentration. Thegrowth eventually plateaued, presumably due to either limited oxygenavailability, since all other essential nutrients were available, orinhibition caused by accumulation of carbon dioxide in the medium orboth. At this point, the culture medium was supplemented with gasvesicles of the present invention such that the culture medium containedabout 1.8% (v/v) crosslinked gas vesicles. As can be viewed in FIG. 8,after the addition of gas vesicles, indicated by the arrow, the cellconcentration increased at least 20%. This demonstrates the improvedoxygen and carbon dioxide carrying capacity of gas-vesicle-supplementedmedia. The biological compatibility of the gas vesicles of the presentinvention should also be appreciated.

Therefore, it is contemplated that gas vesicles can be used to deliver agas to a site or to remove a gas from a site, whether the site isanimate or inanimate matter. As long as gas vesicles can be moved to anenvironment whereby the gas inside the vesicle can diffuse out or thegas outside the vesicle can diffuse in, delivery or removal of a gas canbe achieved. For example, the present invention can be used to deliveroxygen to and/or remove carbon dioxide from, cells—whether those cellsare of the same type or different, as in the case of tissues ororgans—either in vitro or in vivo. It is envisioned that this willtypically be done by putting gas vesicles into a liquid medium, allowingthe oxygen to diffuse from the vesicles into a liquid medium and/orallowing the carbon dioxide to diffuse from the medium into thevesicles, and allowing the oxygen-enriched, carbon dioxide-depletedmedium to flow over the cells and effect gas transfer. However, it isalso envisioned that gas can be removed from a site via gaseous (e.g.,air) or solid media, or possibly even by direct contact with the site,without an intermediate transferring medium.

It is further contemplated that the gas vesicles of the presentinvention can be used to treat mammalian cancers or tumors inconjunction with radiation therapy. Species of tumors exist where, dueto rapid growth, localized hypoxic (oxygen depleted) zones exist. Thesehypoxic cells are not amenable to traditional radiation therapy withoutendangering healthy tissue. Radiation therapy has been shown to be moreeffective by increasing the local oxygen partial pressure around thetumor area. It is envisioned that the injection of gas vesicles canaccomplish this.

Furthermore, gas vesicles may be used as contrast agents for diagnosticultrasound. Diagnostic ultrasound is a tool to gain insight into thestate of internal organs of the body, but its use remains limited in theimaging of certain metastatic lesions. The use of a contrast agent toimprove the imaging has been proposed. Gas vesicles may replacepolymer-coated microbubbles because polymer coated microbubbles arelarger in size. For such a use, the concentration of gas vesicles needonly be about 0.1% (v/v), or possibly as low as 0.01%, to be effective.

Thus it should be evident that the device and methods of the presentinvention are highly effective in obtaining naturally occurring gasvesicles and employing them in biological or medical applications. Theinvention is particularly suited for oxygen delivery and carbon dioxideremoval, but is not necessarily limited thereto. The device and methodof the present invention can be used separately with other equipment,methods and the like.

The present invention also relates to one or more methods of using thesemi-synthetic vesicles of the present invention as contrast agents forultrasonic imaging. In one embodiment, the vesicles are injected intoone or more regions to be imaged. As such, any one or more regions arerendered capable of more efficiently reflecting ultrasonic waves.Accordingly, the one or more regions are more receptive to imaging byultrasound. In one embodiment, vesicles of the present invention can beinjected into a tissue or an organ such as a heart. The vesicles remainin the vicinity of and/or associate with the organ such that the sum ofthe ultrasonic reflections from the vesicles corresponds to the topologyof the organ, or a portion of the organ.

It is well understood in the art that ultrasonic image contrast agentsoperate according to differences in density. More specifically, if animage contrast agent has a density that is sufficiently different fromthe surrounding medium then it is capable of causing acoustic wavereflections. For instance, an acoustic wave propagating through a mediumsuch as biological tissues will be reflected by the less dense agentwhen it interacts with the interface between the medium and the agent.According to the present invention the vesicle contrast agent containsone or more gas-phase components. Thus, there is a large difference indensity between the vesicles' contents and the surrounding tissue media.Therefore, this interface scatters acoustic waves more efficiently thanthe interface between adjacent tissues.

In one embodiment, the vesicles of the present invention are injectedoutside of the organ so that they coat the organ, or some portionthereof. In another embodiment, the vesicles are injected into thetissues of the organ, so that they spread throughout such tissues orsome portion thereof. In still another embodiment, the vesicles areinjected into one or more void spaces within the organ, so that theydiffuse throughout the void space rendering it more susceptible toultrasonic imaging. Furthermore, embodiments of the present inventioncan include any combination of the foregoing injection methods.

The present invention can be used to enhance the ultrasonic image of anyorgan or tissue, human or otherwise. Furthermore, the present inventionis capable of providing image contrast to non-biological objects orsystems.

In another embodiment, the vesicles are chemically bound to a targetingagent, which delivers the vesicles to one or more regions to betargeted. Thus, the vesicle/targeting agent complex can be injected atany point on a body and it will localize in one or more of the targetedregions. For instance, in one embodiment the targeting agent is a bloodplatelet. Thus, one or more vesicles is chemically bound to at least oneplatelet and introduced into an organism's blood stream. The plateletthen localizes in the area of a blood clot. Thus, the blood clot canmore efficiently reflect ultrasonic waves, and can therefore be morereadily imaged ultrasonically.

It is, therefore, to be understood that any variations evident fallwithin the scope of the claimed invention and thus, the selection ofspecific component elements can be determined without departing from thespirit of the invention herein disclosed and described. Thus, the scopeof the invention shall include all modifications and variations that mayfall within the scope of the attached claims.

I claim:
 1. A process of using naturally occurring gas vesicles as acontrasting agent, comprising: collecting cells having naturallyoccurring gas vesicles contained therein; removing the naturallyoccurring gas vesicles from the cells; injecting the naturally occurringgas vesicles in the vicinity of a biological target, wherein thevesicles associate with the target and are operable to reflectultrasonic acoustic waves; subjecting the target to ultrasonic acousticwaves; collecting at least a portion of the reflected ultrasonic waves;converting the collected ultrasonic waves to an electrical signal;transmitting the electrical signal to a computer, wherein the computeris programmed to convert the transmitted electrical signal into an imageof the target; and displaying the image.
 2. The process of claim 1,wherein the step of injecting includes injecting the vesicles into aregion selected from the exterior of a tissue, the interior of a tissue,a void space within the tissue, or any combination thereof.
 3. Theprocess of claim 1 wherein the biological target comprises one or moreof a tissue, an organ, a fetus, a foreign object, or any combinationthereof.
 4. The process of claim 1, wherein the naturally occurring gasvesicles further comprise a targeting agent, wherein the agent directsthe vesicles to the biological target without regard to the proximity ofthe injection site to the biological target.
 5. The process of claim 1,wherein the cells are selected from the group consisting of one or moreof procaryotes, one or more phototropic bacteria, one or morenon-phototropic bacteria, one or more archaea, or any combinationthereof.
 6. The process of claim 5, wherein the one or more procaryotesare selected from one or more cyanobacteria.
 7. The process of claim 6,wherein the one or more cyanobacteria are selected from Microcystisaeruginosa, Aphanizomenon flos aquae and Oscillatoria agardhii.
 8. Theprocess of claim 5, wherein the one or more phototropic bacteria areselected from Amoebobacter, Thiodictyon, Pelodictyon, and Ancalochloris.9. The process of claim 5, wherein the one or more non-phototropicbacteria are selected from Microcyclus aquaticus.
 10. The process ofclaim 5, wherein the one or more archaea are selected from Haloferaxmediterranei, Methanosarcina barkeri, and Halobacteria salinarium.